PROJECTSUMMARY Acute respiratory distress syndrome (ARDS) is a major cause of mortality in critically ill patients in the US. PharmacologictherapiesforARDShavebeenlargelyunsuccessfulindecreasingmortality.Patientssufferfrom severe hypoxemia caused by acute inflammation, lung epithelial and endothelial cell damage, and capillary leakage of protein-rich fluid into alveoli. Studies from our laboratory have shown that dysregulation of protein degradationalterslunginflammationinexperimentalmodelsofARDS.Thelong-termgoalofourresearchisto identifytherapeutictargetstodecreaseinflammationinARDSandreducemortality.Theubiquitin-proteasome pathway (UPP) is responsible for most protein degradation in cells. This irreversible process is critical to maintaining cell homeostasis. Failure to regulate protein stability can lead to tissue damage from unchecked inflammation. Glycogen synthase kinase-3? (GSK3?) is a highly conserved serine-threonine kinase and key regulatorofcelldifferentiation,metabolism,andinflammation.PhosphorylationofkeyproteinsbyGSK3?has manybiologicalfunctions,oneofwhichistargetingsubstratesfordegradationbytheUPP.Studieshaveshown thatGSK3?mediatesdeleteriouslunginflammationinmurinemodelsofsepsisandARDS.Despiteitscritical roleincellbiology,littleisknownabouthowGSK3?stabilityitselfisregulated,particularlyinthecontextoflung inflammation.Thegoalsofourprojectare1)todefinemechanismsofGSK3?degradationinlungepithelialcells;? 2) to determine how inflammation affects GSK3? kinase activity and protein stability in pathways relevant in ARDS;? and 3) to identify novel E3 ligases that regulate GSK3? stability in models of ARDS. Using a cycloheximidechaseassaytostudythelifespanofGSK3?,wewilloverexpressmutantGSK3?inmurinelung epithelial(MLE)cellstodeterminewhichlysineresiduesandstructuralmotifsarerequiredfordegradation.We willalsouseinflammatorystimuliincludingLPSandTNFa?andoverexpressmutantformsofGSK3?todetermine howGSK3?kinaseactivityaltersitsstabilityinMLEcells.Finally,wewillscreenalibraryofF-boxproteinsto identify a novel E3 ubiquitin ligase complex that degrades GSK3? and study the effects on inflammatory responsesinlungepithelialcells.Thisworkwillelucidatepotentialtargetsfornoveltherapiestoreducemortality inARDS.AsapulmonaryandcriticalcaremedicinefellowintheAcuteLungInjuryCenterofExcellenceatthe UniversityofPittsburgh,sheismentoredbyDrs.RamaMallampalliandYutongZhaotobecomeproficientin laboratory models of acute lung injury. As a postdoctoral research fellow her training will focus on advanced laboratorytechniquesinbiochemistry,molecularandcellularbiology,andmolecularimmunology.Earlycareer developmentopportunitiesincludedidactictraining,conferenceattendanceandpresentations,andpublications andthatwillbeguidedthroughmentoringbyherco-sponsors.ThesupportoftheNationalResearchService Awardforthisproposalwillserveasthefoundationforaccomplishinghergoalofbecominganindependentand successfulphysician-scientist.